Intraocular neuroectodermal embryonal tumor in two rabbits.

Spontaneous intraocular tumors are rarely reported in rabbits, despite their widespread use as laboratory animals. We describe two cases of intraocular neuroectodermal embryonal tumors, formerly primitive neuroectodermal tumors, in young rabbits. Histologically, both tumors exhibited prominent rosette or pseudorosettes, consistent with the histomorphology seen in human tumors. The neuroectodermal subtype is supported by immunoreactivity for the neuronal markers, SRY-box transcription factor 2, microtubule-associated protein 2, neuronal nuclear protein, and neuron-specific enolase. In one of the rabbits, there was metastasis to the contralateral conjunctiva. Intraocular neoplasms can occur in young rabbits and eyes with refractory disease should be enucleated for clinical management.

Pathology in Practice.

In collaboration with the American College of Veterinary Pathologists.

Pulmonary and systemic responses to aerosolized lysate of Staphylococcus aureus and Escherichia coli in calves.

BACKGROUND: Constitutive and inducible defenses protect the respiratory tract from bacterial infection. The objective of this study was to characterize the response to an aerosolized lysate of killed bacteria, as a basis for studying the regulation and in vivo effects of these inducible innate immune responses. RESULTS: Bacterial lysate consisting of heat-killed and sonicated Staphylococcus aureus and Escherichia coli was aerosolized to 6 calves and systemic and pulmonary innate immune and inflammatory responses were measured in the first 24 h relative to baseline. Evaluated parameters included clinical parameters (body temperature and heart and respiratory rates), blood acute phase proteins and leukocyte counts, and leukocytes and proteins in bronchoalveolar lavage fluid. Mild clinical signs with increased heart rates and rectal temperatures developed following administration of the lysate, with resolution by 24 h. Serum haptoglobin and plasma fibrinogen concentrations were elevated at 24 h relative to baseline. Bronchoalveolar lavage fluid (BALF) had increased cellularity and increased proportion of neutrophils, as well as higher concentrations of interleukin (IL)-8, IL-10 and total protein at 24 h relative to baseline. Mass spectrometry identified 965 unique proteins in BALF: 19 proteins were increased and 26 proteins were decreased relative to baseline. The upregulated proteins included those involved in innate immunity including activation of complement, neutrophils and platelets. At postmortem examination, calves receiving higher doses of lysate had areas of lobular consolidation and interlobular edema. Histologically, neutrophils were present within bronchioles and to a lesser extent within alveoli. Calves receiving highest doses of lysate had patchy areas of neutrophils, hemorrhage and hyaline membranes within alveoli. CONCLUSIONS: Aerosolization of bacterial lysate stimulated an innate immune response in lungs and airways, with alveolar damage observed at higher doses. Such a stimulus could be of value for investigating the effects of inducible innate immune responses on occurrence of disease, or for evaluating how stress, drugs or genetics affect these dynamic responses of the respiratory tract.

Benzo(a)pyrene suppresses tracheal antimicrobial peptide gene expression in bovine tracheal epithelial cells.

Respiratory disease is an important cause of morbidity and mortality in cetaceans, which are also threatened by environmental degradation caused by crude oil spills. Following oil spills, cetaceans at the water surface may inhale droplets of oil containing toxic polycyclic aromatic hydrocarbons (PAHs), which could potentially alter respiratory immunity via activation of the aryl hydrocarbon receptor (AHR) and its subsequent interaction with nuclear factor kappa B (NF-κB). ß-defensins are antimicrobial peptides secreted by airway epithelial cells and their expression is known to be dependent on NF-κB. We hypothesized that PAHs may suppress the expression of ß-defensins, and thereby contribute to the pathogenesis of pneumonia. This hypothesis was modeled by measuring the in vitro effects of benzo(a)pyrene (BAP), phenanthrene, and naphthalene on tracheal antimicrobial peptide (TAP) gene expression in bovine tracheal epithelial cells. Stimulation with lipopolysaccharide (LPS) induced 20 ± 17-fold (mean ± SD) increased TAP gene expression. Exposure of tracheal epithelial cells to 5 µM BAP for 4 or 8 h, followed by incubation with a combination of LPS and 5 µM BAP for another 16 h, significantly (P = 0.002) suppressed LPS-induced TAP gene expression by 40.6 ± 21.8% (mean ± SD) in tracheal epithelial cells from 9 calves tested. BAP-induced suppression of TAP gene expression coincided with induction of cytochrome P450 1A1 gene expression. In contrast, phenanthrene and naphthalene had no consistent effect, and exposure to PAHs did not significantly affect constitutive TAP gene expression (i.e. without LPS). These findings characterize the suppressive effects of BAP-a toxic pollutant found in crude oil-on this respiratory innate immune response.

Response to Intravenous Allogeneic Equine Cord Blood-Derived Mesenchymal Stromal Cells Administered from Chilled or Frozen State in Serum and Protein-Free Media.

Equine mesenchymal stromal cells (MSC) are commonly transported, chilled or frozen, to veterinary clinics. These MSC must remain viable and minimally affected by culture, transport, or injection processes. The safety of two carrier solutions developed for optimal viability and excipient use were evaluated in ponies, with and without allogeneic cord blood-derived (CB) MSC. We hypothesized that neither the carrier solutions nor CB-MSC would elicit measurable changes in clinical, hematological, or biochemical parameters. In nine ponies (study 1), a bolus of HypoThermosol(®) FRS (HTS-FRS), CryoStor(®) CS10 (CS10), or saline was injected IV (n = 3/treatment). Study 2, following a 1-week washout period, 5 × 10(7) pooled allogeneic CB-MSCs were administered IV in HTS-FRS following 24 h simulated chilled transport. Study 3, following another 1-week washout period 5 × 10(7) pooled allogeneic CB-MSCs were administered IV in CS10 immediately after thawing. Nine ponies received CB-MSCs in study 2 and 3, and three ponies received the cell carrier media without cells. CB-MSCs were pooled in equal numbers from five unrelated donors. In all studies, ponies were monitored with physical examination, and blood collection for 7 days following injection. CD4 and CD8 lymphocyte populations were also evaluated in each blood sample. In all three studies, physical exam, complete blood cell count, serum biochemistry, and coagulation panel did not deviate from established normal ranges. Proportions of CD4(+) and CD8(+) lymphocytes increased at 168 h postinjection in CB-MSC treatment groups regardless of the carrier solution. Decreases in CD4(+/)CD8(+) double positive populations were observed at 24 and 72 h in CB-MSC-treated animals. There was no difference in viability between CB-MSCs suspended in HTS-FRS and CS10. HTS-FRS and CS10 used for low volume excipient injection of MSC suspensions were not associated with short-term adverse reactions. HTS-FRS and CS10 both adequately maintain CB-MSC viability following hypothermic or frozen simulated transport, respectively. CB-MSCs do not elicit clinical abnormalities, but allogeneic stimulation of CD4(+) and CD8(+) lymphocyte populations may occur. Future studies should include in vitro or in vivo evaluation of cell-mediated or adaptive immunity to autologous, identical allogeneic, or MSC originating from additional unrelated individuals in order to better characterize this response.

PIWIL1 Is Expressed in the Canine Testis, Increases with Sexual Maturity, and Binds Small RNAs.

Spermatogenesis is a highly regulated process leading to the development of functional spermatozoa through meiotic division and subsequent maturation. Recent studies have suggested that a novel class of Argonaute proteins, known as the PIWI clade, plays important roles in multiple stages of spermatogenesis. PIWI proteins bind specific small noncoding RNAs, called PIWI-interacting RNAs (piRNAs). These piRNAs guide the PIWI-piRNA complex to retrotransposon targets that become expressed during meiosis. Retrotransposons are subsequently silenced, either through PIWI "slicer" activity or through PIWI-directed methylation of the retrotransposon locus. Most mammalian studies have employed mouse models where sterility follows PIWI inactivation. The goal of this study was to characterize canine PIWIL1 to determine whether expression pattern and functional characteristics support a similar function in that species. Canine PIWIL1 cDNA is a 2.6-kb transcript that encodes an 861-amino acid protein showing high homology to other mammalian PIWIL1 proteins and containing features consistent with PIWI family members (PAZ, PIWI domains). Analysis of PIWIL1 protein and transcript levels revealed that PIWIL1 expression is limited to the testes and is associated with sexual maturity, with mature dogs showing higher levels of PIWIL1 expression. Immunohistochemistry demonstrated expression primarily in seminiferous tubules and confirmed higher levels of PIWIL1 in mature dogs. Functional characterization by RNA immunoprecipitation demonstrated that canine PIWIL1 binds short RNAs consistent in size with piRNAs (27-32 nucleotides). Together, these studies represent the first characterization of a PIWI protein in the dog and suggest that it is a functional piRNA-binding protein most highly expressed in the mature testes.

Canine Platelet Lysate Is Inferior to Fetal Bovine Serum for the Isolation and Propagation of Canine Adipose Tissue- and Bone Marrow-Derived Mesenchymal Stromal Cells.

BACKGROUND: Mesenchymal stromal cells (MSC) are increasingly investigated for their clinical utility in dogs. Fetal bovine serum (FBS) is a common culture supplement used for canine MSC expansion. However, FBS content is variable, its clinical use carries risk of an immune response, and its cost is increasing due to global demand. Platelet lysate (PL) has proven to be a suitable alternative to FBS for expansion of human MSC. HYPOTHESIS AND OBJECTIVES: We hypothesized that canine adipose tissue (AT) and bone marrow (BM) MSC could be isolated and expanded equally in PL and FBS at conventionally-used concentrations with differentiation of these MSC unaffected by choice of supplement. Our objectives were to evaluate the use of canine PL in comparison with FBS at four stages: 1) isolation, 2) proliferation, 3) spontaneous differentiation, and 4) directed differentiation. RESULTS: 1) Medium with 10% PL was unable to isolate MSC. 2) MSC, initially isolated in FBS-supplemented media, followed a dose-dependent response with no significant difference between PL and FBS cultures at up to 20% (AT) or 30% (BM) enrichment. Beyond these respective peaks, proliferation fell in PL cultures only, while a continued dose-dependent proliferation response was noted in FBS cultures. 3) Further investigation indicated PL expansion culture was inducing spontaneous adipogenesis in concentrations as low as 10% and as early as 4 days in culture. 4) MSC isolated in FBS, but expanded in either FBS or PL, maintained ability to undergo directed adipogenesis and osteogenesis, but not chondrogenesis. CONCLUSIONS/SIGNIFICANCE: Canine PL did not support establishment of MSC colonies from AT and BM, nor expansion of MSC, which appear to undergo spontaneous adipogenesis in response to PL exposure. In vivo studies are warranted to determine if concurrent use of MSC with any platelet-derived products such as platelet-rich plasma are associated with synergistic, neutral or antagonistic effects.